Genome Organization of Escherichia Phage YD-2008.s: A New Entry to Siphoviridae Family.

Malaysia is one of the countries that are loaded with mega biodiversity which includes microbial communities. Phages constitute the major component in the microbial communities and yet the numbers of discovered phages are just a minute fraction of its population in the biosphere. Taking into account of a huge numbers of waiting to be discovered phages, a new bacteriophage designated as Escherichia phage YD-2008.s was successfully isolated using Escherichia coli ATCC 11303 as the host. Phage YD-2008.s poses icosahedral head measured at 57nm in diameter with a long non-contractile flexible tail measured at 107nm; proving the phage as one of the members of Siphoviridae family under the order of Caudovirales. Genomic sequence analyses revealed phage YD-2008.s genome as linear dsDNA of 44,613 base pairs with 54.6% G+C content. Sixty-two open reading frames (ORFs) were identified on phage YD-2008.s full genome, using bioinformatics annotation software; Rapid Annotation using Subsystem Technology (RAST). Among the ORFs, twenty-eight of them code for functional proteins. Thirty two are classified as hypothetical proteins and there are two unidentified proteins. Even though majority of the coded putative proteins have high amino acids similarities to phages from the genus Hk578likevirus of the Siphoviridae family, yet phage YD-2008.s stands with its' own distinctiveness. Therefore, this is another new finding to Siphoviridae family as well as to the growing list of viruses in International Committee on Taxonomy of Viruses (ICTV) database.

Complete Genome Sequence of Proteus mirabilis Phage pPM_01 Isolated from Raw Sewage.

OBJECTIVES: Phage pPM_01 was previously isolated from a raw sewage treatment facility located in Batu Maung, Penang, Malaysia, and it was highly lytic against Proteus mirabilis, which causes urinary tract infections in humans. In this paper, we characterize the biology and complete genome sequence of the phage. METHODS AND RESULTS: Transmission electron microscopy revealed phage pPM_01 to be a siphovirus (the first reported virus to infect P. mirabilis), with its complete genome sequence successfully determined. The genome was sequenced using Illumina technology and the reads obtained were assembled using CLC Genomic Workbench v.7.0.3. The whole genome contains a total of 58,546 bp of linear double-stranded DNA with a G+C content of 46.9%. Seventy putative genes were identified and annotated using various bioinformatics tools including RAST, Geneious v.R7, National Center for Biotechnology Information (NCBI) BLAST, and tRNAscan-SE-v1.3 Search. Functional clusters of related potential genes were defined (structural, lytic, packaging, replication, modification, and modulatory). The whole genome sequence showed a low similarity to known phages (i.e., Enterobacter phage Enc34 and Enterobacteria phage Chi). Host range determination and SDS-PAGE analysis were also performed. CONCLUSIONS: The inability to lysogenize a host, the absence of endotoxin genes in the annotated genome, and the lytic behavior suggest phage pPM_01 as a possible safe biological candidate to control P. mirabilis infection.

PCR assembly of synthetic human erythropoietin gene

Human erythropoietin (huEPO) is a glycoprotein with important physiological functions, such as erythropoiesis, angiogenesis, and wound healing. A therapeutic protein, huEPO is commonly used to treat patients suffering from renal and non-renal anemia. Recombinant human erythropoietin (rhuEPO) and endogenous huEPO are similar with respect to their biological and chemical properties. In this study, we describe the construction of synthetic huEPO gene to produce rhuEPO. The synthetic huEPO gene was constructed by overlapping oligonucleotides assembly and amplified by polymerase chain reaction (PCR). Twenty oligonucleotide sets, covering the huEPO gene sequence and two newly introduced restriction enzyme sites, were pulled together and amplified using Pfu DNA polymerase to produce the expected DNA products with sizes of ~500bp and ~600bp. The PCR products were ligated into pGEM-T plasmid vector to facilitate DNA sequencing process of the constructed huEPO gene and downstream cloning manipulation. DNA sequence analysis showed correctly assembled oligonucleotide sets, representing the huEPO gene sequence albeit with minor base mutations. Hence, oligonucleotides assembly and PCR amplification provide a convenient and speedy method for the synthesis of huEPO gene without depending on mRNA isolation and reverse transcription or the need to have a genomic library.